The lactone of 2-oxo-6-(2-hydroxypropyl)-cyclohexanecarboxylic acid



United States Patent Ofilice 3,079,401 Patented Feb. 26, 1963 3,079,401THE LACTDNE 9F 2@XG--(2-HYDRXYPRPYL)- CYCLOHEXANECARBSXYLEC ACID ChesterR. Benjamin, Hyattsville, Md, and William F.

Hendershot and Clifford W. Hesseltine, Peoria, El, assignors to theUnited States of America as represented by the Secretary of AgricultureNo Drawing. Original application Sept. 18, 1961, Ser. No. 139,004, newPatent No. 3,063,909, dated Nov. 13, 1962. Divided and this applicationMay 17, 1962, Ser. No. 202,342

1 Claim. (Cl. ass-343.2 (Granted under Titie 35, US. Code (1952), see.266) This application is a division of copending application Serial No.139,004, filed September 18, 1961, now US. Patent No. 3,063,909.

A nonexclusive, irrevocable, royalty-free license in the inventionherein described, throughout the World for all purposes of the UnitedStates Government, with the power to grant sublicenses for suchpurposes, is hereby granted to the Government of the United States ofAmerica.

This invention relates to a novel seed and fungal spore germinationinhibitor obtained in crystalline form from certain unusualfermentations of a nonsporulating strain of microorganisms, namelyPestalotia ramulosa v. Beyma, NRRL 2826.

More particularly this invention relates to the discovery that underhighly unobvious low temperature conditions of culture or fermentationPestalotia ramulosa, NRRL 2826 reproduces without forming spores andthat at an optimal temperature of only C. the mycelium of this strainforms large amounts of a novel crystalline compound, solutions ordispersions of which, we discovered, are able to selectively inhibit thegermination of grass and plant seeds and the spores of a variety offungi. The crystalline compound produced in the specific cultures andfermentations of Pestalotia ramzzlosa NRRL 2826 under the atypical andhighly critical fermentation conditions described below has beendetermined to have the empirical formula c mp, that may be chemicallydesignated as the lactone of2-oxo-6-(2-hydroxypropyl)-cyclohexanecarboxylic acid. In recognition ofits source, we call the above novel compound ramulosin.

Three varieties or strains of Pestalotza ramulosa are known, namelyPestalotia (T rzmcatella) ramulosa v. Beyma, NRRL 2826, P. ramulosa NRRL1228, and P. ramulosa NRRL A-9471. The germinative forms of the threevarieties are indistinguishable from one another even under themicroscope, but carbon and nitrogen utilization studies employingextensive spectra of carbohydrates as shown in Table I and amino acidsas shown in Table II indicate distinguishing differences in utilizationand, most importantly, marked differences as to the presence or absenceof the ability to sporulate. Under the condi-v tions of the abovestudies P. ramzzlosa NRRL 2826 did not sporulate with any of the carbonand nitrogen sources; P. ramulosa NRRL 1228 formed spores with 8 of the16 carbon sources and with all nine of the nitrogen sources; P. ramulosaNRRL A-947l formed spores with 14 of the 16 carbon sources as well as inthe absence of a carbon source, and formed spores in the presence of 8of the 9 nitrogen sources as well as in the absence of a nitrogensource.

In furtherance of our unexpected discovery of some macroscopic colorlessto white crystals of ramulosin in a malt extract agar culture of P.ramulosa NRRL 2826, the following objects of our invention havedeveloped. A principal object is the discovery of a novel compound offungal origin. Another object is the discovery of culture andfermentation conditions that provide high yields of the hitherto unknowncompound. A still further object is the discovery of a microbiologicalproduct having distinctly inhibitory effects on fungi andanti-germination activity on plant seeds including grass, and vegetableseeds. The above and other objects of our invention will become clear inthe course of the following specification.

In accordance with the present invention we have now unexpectedlydiscovered that the mycelium of Pestalotia ramulosa v. Beyma NRRL 2826,an imperfect fungus belonging to the Order Melanconiales, producesmaximum yields of ramulosin in fermentations or cultures maintained at15 0, moderate yields at up to 25 C., only traces at 28 C. and no yieldof ramulosin above 28 C.

We have discovered that Pestalotia ramulosa NRRL 2826 never forms sporesbut produces ramulosin, whereas scarcely distinguishable P. ramulosaNRRL 1228 and P. ramulosa NRRL A-9471 do sporulate under certainordinary conditions but produce no ramulosin. From these minimalobservations we theorized that perhaps the ramulosin (which is producedonly in the consistently nonsporulating strain) may be responsibleforand associated with the absence of sporulation in P. ramulosa NRRL 2826and, by extension, we considered whether ramulosin might not alsoinhibit the germination of other plant and vegetable life. We have nowverified the above hypothesis.

Our first discovery of ramulosin crystals, as already indicated, wasnoted in a profuse mycelial growth on a malt extract agar plate. Thismode of propagation is, of course, wholly unsuitable for any large-scaleproduction. We subsequently found that the best fermentation medium forproducing ramulosin comprises 4.0 percent malt extract, 4.0 percentdextrose, and 0.1 percent peptone, and that ramulosin is produced bothin submerged and in surface fermentations conducted at 15 (optimal) to25 C. during the 5-9 weeks succeeding an unproductive initial period of2-4 weeks in which the growing mycelium must apparently firstsubstantially exhaust the carbon source present in the fermentationmedium and perhaps also adapt to some inhibitory metabolite or to anintermediate which might then be further changed into the actualinsoluble crystals of ramulosin.

At the end of the fermentation, pure ramulosin was obtained byextracting the filterable solids two times with 2 volumes of acetone andthen twice with ether, recovering each extract by filtration, addingthereto the 12 hour ether extract of a tenfold vacuum concentrate of theculture filtrate, evaporating to dryness to obtain a crude product,successively recrystallizing the ramulosin from aqueous acetone, thenfrom hexane, and again from aqueous acetone, filtering, and air-dryingto obtain crystalline ramulosin melting at 121.0205 C. Ramulosin has arotation of [a] +17.6 (e. 2.9; ethanol). Under UV it shows a single peakat 264 mu (e=10,100). It shows strong IR bands respectively at 6.05,8.05, and 11.2 ,u. The benzoate (C I-1 melted at 80-81 C. The4-dimethylamino-3,5-dinitrobenzoate (C H O N melted at 179-181 C. Thep-phenylazobenzoate (C H O N melted at 133-134 C.

in a standard seed germination tests (Rules and Regulations Under theFederal Seed Act, USDA, 1956, p. 29) ramulosin was solubilized atconcentrations of 1, 10, 100, 1000 ppm. in boiling water containing 1percent of a commercially available surface active polyoxyethyleuederivative of fatty acid partial esters of hexitol anhydride althoughany other surface active dispersing agent would do. Seeds were soaked inthe cooled solution for one hour before incubating in letri dishes.Tomato and grass seeds were incubated at room temperature and eX- posedto the natural diurnal cycle. The other seeds were incubated in the darkat 28 C. The results are shown in Table III.

Ramulosin was also tested for its efiect on various fungi. At 1000 ppm.it retarded but did not prevent the mycelial growth of a spore-formingstrain of P. ramulosa. At 250 ppm. it prevented germination on syntheticmucor agar (Hesseltine, Mycologia 46, 358, 1554) of conidiospores ofAspergillns niger NRRL 3 and of Fusarium monilijorme NRRL 2374. It alsoinhibited the germination of ascospores of Chaeromium globosnm NRRL 1870and of sporangiospores of Rlzizopns stolonifer NRRL 2233 and of Mncorrouxii NRRL 1894. At 1000 ppm. ramulosin inhibited proliferation of thebasidiomycete, Ustilago maydis NRRL 2321. These results are summar-izedin Table IV.

The following specific examples show that ramulosin is produced both insurface and in submerged fermentations. Although for convenience weusually employed malt extract, glucose, and peptone as the carbon andnitrogen sources, D-glutamic acid is the best source of nitrogen, andthe data of Tables I and i1 suggest other sources that might besubstituted.

Also, since the production of ramulosin does not begin until there hasbeen an extensive proliferation of mycelia, commercially it would beadvantageous to speed the growth of the mycelia by initially fermentingat 25- 28 C., until the carbon source is virtually exhausted, and thenlowering the temperature to about 15 C. to induce the maximum productionof ratnulosin by the mycelia.

EXAMPLE 1 (Surface Fermentation) A 1 sq. cm. block of malt-extract agarmedium which had been inoculated by a loop transfer of stock from aculture of P. ramulosa NRRL 2826 and incubated at 25 C. for 47 days wastransferred to 300 ml. of malt-extract broth in a 2800 ml. Fernbachilask and fermented as a still culture held at 15 C. for days. Themalt-extract broth fermentation medium consisted of 40 g. of maltextract, 1 g. of peptone, and 40 g. of D-glucose in 1000 ml. distilledwater. The original pH was 5.4 and the final pH was 4.3. Ramulosincrystals were first seen at 26 days. At 66 days the dry weight ofmycelia was 2.08 g. per 1000 ml. of fermentation medium and the yield oframulosin was 1.45 gm. At 68 days the yield of crystalline ramulosin was1.50 gm. per 1000 ml. compared with 1.24 gm. from a parallelfermentation at 20 C. and of only 0.98 gm. per 1000 ml. from afermentation at 25 C. A 90 day fermentation at 15 C. yielded 6.2 gm. perliter of ramulosin, equivalent to 15.8 percent of the glucose added. Afermentation at 23 C. produced only a trace of mycelial growth and noramulosin.

EXAMPLE 2 (Surface Fermentation) Fermentations similar to those ofExample 1 were carried out at 15 C. in a less concentrated mediumcontaining malt extract 20 g., peptone 1 g., and D-glucose 20 g. per1000 ml. of distilled water. The fermentation was harvested at 90 daysand 2.8 gm. of crystalline rarnulosin per 1000 ml. of fermentationmedium was obtained.

EXAMPLE 3 (Submerged Shaken Fermentation) A fermentation similar to thatof Example I was incubated on a rotary shaker at 15 C. for 74 days.Although the mycelium apparently agglutinated and rolled into a largeclump (5.0 gm. per 1000 ml.) which interfeted with its oxidativemetabolism, a suboptimal yield of 3.0 gm. of ramulosin per 1000 ml. ofmedium (equivalent to 7.5 percent of the glucose added to thefermentation) was obtained.

EXAMPLE 4 (Submerged Still Fermentation) A pilot plant scalefermentation of 200 gal. of maltextract broth (4 percent glucose, 4percent malt extract and 0.1 percent peptone) was inoculated with 20gal. of the culture grown on a similar medium for 7 days at 25 C. Thefermentation was run at 18 C. for 28 days and yielded a total of 1125gms. of pure rarnulosin or 5.1 gm. per liter.

TABLE I. UTILIZATION OF CARBON COMPOUNDS BY STRAINS OF PESTALOTIARAMULOSA Utilization Sporulation NRRL NRRL NRRL NRRL NRRL NRRL 2826 122811-9471 282 I228 A-Qi'll s1. s1. i

s1. sl. 51. s1. t t t t TABLE II. UTILIZATION OF NITROGEN COMPOUNDS BYSTRAINS OF PES'IALOTIA RAMULOSA Utilization Sporulation NRRL NRRL NRRLNRRL NRRL NRRL 2826 1228 A-9471 2826 1228 A-9471 -Prohue.

TABLE III. EFFECT OF RAMULOSIN ON SEED GERMINATION Percent of seedsgerminated Incubation Seed time, Controls P.p.m. of ramulosin days WaterTween-80 1.000 100 1 1 Standard Trocedures were followed. B Theseseedlings were stunted as compared with the control seedlings.

TABLE IV. EFFECT OF RAMULOSIN' ON GERMINATION OF SELECTED FUNG-AL SPORESRamulosin (p.p.m.) Culture Spore type Incubation time. hrs.

Percent inhibition 9 Aspergz'll'us m'gcr N RRL 3. Conidiospores 24 0 0 048 0 0 0 (20) Fusarium monilijorme .(lo 24 0 0 (16) 100 NRRL 2374. 48 00 0 (20) Ohaetomium globosum 3 Ascospores 24 0 N RRL 1870. 48 0 65 65100 Rhizopus stolonifer NRRL Sporangiospores-.- 24 0 0 0 100 2233. 48 00 0 71 Mucor rouzii NRRL 1894. -do 24 0 0 0 100 48 0 0 0 (10)N 1Incubation time is the number of hours after treatment with ramulosin.

Controls with the equivalent amount of alcohol as in the tests were run.Numbers in parentheses indicate retarded growth.

a This culture germinates slowly and no counts were made at 24 hours.

Having fully disclosed our invention, We claim: The lactone of 2 0X0 6(2 hydroxypropyl) cyclohexanecarboxylic acid.

References Cited in the file of this patent Benjamin et a1.: Nature,188, pages 662-663 (Nov. 19, 1960), QLNZ.

